There is a need for simple, scalable methods for immobilization of proteins on a variety of materials for many applications e.g. diagnostic biosensors, immunodiagnostics, surgical implants, and bioprocessing media for affinity purification. In all applications it is critical to preserve the protein structure and function, and to orientate it correctly on the substrate for maximum efficiency of performance. There are numerous strategies for the immobilization of proteins on surfaces, as reviewed in Nakanishi, K. et al. ((2008) Current Proteomics 5: 161-175).
Physisorption is a commonly used method based on hydrophobic or ionic interactions of the protein with the surface. It is methodologically simple, but allows little quantitative or orientational control; it may alter the functional properties of the protein through unfolding, and reproducibility and efficiency are variable. This means that when bound, the proteins may not be oriented correctly for analyte binding or may be rendered wholly or partially denatured and non-functional. Many of these methods are complicated with many process steps and difficult to scale for mass manufacture. The methods also result in a large percentage loss of function of the protein concerned; for example less than 10% of antibody adsorbed to plastic ELISA plates is active whilst chemical coupling e.g. amine coupling results in 75-100% loss of activity (Butler J. E. et al. (1992) J Immunol Methods 150: 77-90; Butler J. E. et al. (1992) J Immunol Methods 150: 77-90; Esser, P. (2010) Thermo Scientific Application Note 11b; Johnsson, B. et al. (1995) J Molecular Recognition 8: 125-131).
Other methods of binding proteins to a surface include functionalization of the surface, for example via amination or carboxylation to allow covalent coupling of the proteins to the surface, or coating of the surface to mediate binding (e.g. streptavidin or biotin coating, polyLys coating, protein A coating, or nickel coated surfaces). Other methods include modification of the protein by addition of a binding tag (for example PhaF) which binds to bioplastic PHA derived from bacteria. Such methods based on covalent coupling provide a stable linkage, can be applied to a range of proteins and have good reproducibility. However, orientation may be variable, and chemical derivatisation may alter the function of the protein and requires a stable, interactive surface. Biological capture methods utilising a tag (such as hexahistidine/Ni-NTA or biotin/avidin) on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the partner reagent must first be immobilised adequately on the surface.
WO2002057780 describes the binding of protein to gold surfaces in self-assembled monolayers where the biologically functional moiety of the protein is correctly oriented and retains close to 100% of its activity. The technology involves the use of a modified variant of a β-barrel structured bacterial outer membrane protein (OMP) as a scaffold upon which other proteins and peptides of interest may be fused. The scaffold has intrinsic self-assembling properties on gold and is used as the anchor point for a fusion partner. The fusion partner is correctly oriented and retains function. The binding of the OMP to the gold surface relies upon modification of the protein to include a cysteine residue at an appropriate position in the OMP such that when the cysteine forms a covalent bond with the gold surface the protein is correctly orientated and directly coupled to the surface.
These methods have the disadvantage of requiring specific amino acids in the protein to mediate the reaction with the surface, modifications of the surface to accept covalent bonding from the cysteine, and the presence of amphiphilic molecules such as thiolipids or thioalkanes to stabilise the protein monolayer.
The applications requiring protein attachment to a surface are widespread, often requiring high protein density across a small area in order to maximise assay sensitivity. Plastic has many advantages for use as a substrate in protein based assays. However, the hydrophobic nature of plastic surfaces results in non-specific binding and denaturation of the proteins.
The present invention aims to overcome or ameliorate some of the problems associated with the prior art.